Toxicology Gene expression profiles in rat lung after ...

Viewer
Transcript

Toxicology 258 (2009) 47–55

Contents lists available at ScienceDirect

Toxicology journal homepage: www.elsevier.com/locate/toxicol

Gene expression proﬁles in rat lung after inhalation exposure to C60 fullerene particles Katsuhide Fujita a,∗ , Yasuo Morimoto b , Akira Ogami b , Toshihiko Myojyo b , Isamu Tanaka b , Manabu Shimada c , Wei-Ning Wang c , Shigehisa Endoh d , Kunio Uchida d , Tetsuya Nakazato d , Kazuhiro Yamamoto e , Hiroko Fukui a , Masanori Horie a , Yasukazu Yoshida a , Hitoshi Iwahashi a , Junko Nakanishi f a

Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Onogawa 16-1, Tsukuba, Ibaraki 305-8569, Japan Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan c Graduate School of Engineering, Hiroshima University, Higashi Hiroshima 739-8527, Japan d Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8569, Japan e Research Institute of Instrumentation Frontier, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8568, Japan f Research Institute of Science for Safety and Sustainability, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8569, Japan b

a r t i c l e

i n f o

Article history: Received 20 October 2008 Received in revised form 22 December 2008 Accepted 5 January 2009 Available online 9 January 2009 Keywords: C60 fullerene Nickel oxide Gene expression Lung DNA microarray Nanoparticles

a b s t r a c t Concern over the inﬂuence of nanoparticles on human health has risen due to advances in the development of nanotechnology. We are interested in the inﬂuence of nanoparticles on the pulmonary system at a molecular level. In this study, gene expression proﬁling of the rat lung after whole-body inhalation exposure to C60 fullerene (0.12 mg/m3 ; 4.1 × 104 particles/cm3 , 96 nm diameter) and ultraﬁne nickel oxide (Uf-NiO) particles (0.2 mg/m3 ; 9.2 × 104 particles/cm3 , 59 nm diameter) as a positive control were employed to gain insights into these molecular events. In response to C60 fullerene exposure for 6 h a day, for 4 weeks (5 days a week), C60 fullerene particles were located in alveolar epithelial cells at 3 days post-exposure and engulfed by macrophages at both 3 days and 1 month post-exposures. Gene expression proﬁles revealed that few genes involved in the inﬂammatory response, oxidative stress, apoptosis, and metalloendopeptidase activity were up-regulated at both 3 days and 1 month post-exposure. Only some genes associated with the immune system process, including major histocompatibility complex (MHC)-mediated immunity were up-regulated. These results were signiﬁcantly different from those of Uf-NiO particles which induced high expression of genes associated with chemokines, oxidative stress, and matrix metalloproteinase 12 (Mmp12), suggesting that Uf-NiO particles lead to acute inﬂammation for the inhalation exposure period, and the damaged tissues were repaired in the post-exposure period. We suggest that C60 fullerene might not have a severe pulmonary toxicity under the inhalation exposure condition. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction In recent years, advances in the development of nanotechnology have raised concerns over the inﬂuence of ultraﬁne nanoparticles on human health and the environment. Based on the clinical assessments of exposure to dusts, asbestos, or suspended particulate matters, there has been more interest in the inﬂuences of manufactured ultraﬁne particles on pulmonary inﬂammation, ﬁbrosis, and cancer (Stone et al., 2007). The pulmonary toxicity of rodents exposed to nanoparticles has been reported (Warheit et al., 2004;

∗ Corresponding author. Fax: +81 298 861 8260. E-mail address: [email protected] (K. Fujita). 0300-483X/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2009.01.005

Grassian et al., 2007; Mitchell et al., 2007; Warheit et al., 2007). The use of C60 fullerene, a carbon-based nanoparticle with a spheroidal network structure, is expected to grow in diverse industrial ﬁelds. In spite of interest in the potential toxicological impact of watersoluble C60 fullerene, little is known about its mechanism of action in vivo. It was reported that suspensions of C60 fullerene in water had little or no difference to lung toxicity effects (Sayes et al., 2007). The assessment of toxicity resulting from inhalation exposures to C60 fullerene nanoparticles and microparticles determined minimal changes in the toxicological endpoints (Baker et al., 2008). To discuss the toxicity of C60 fullerene, further information regarding the in vivo mechanisms is still needed. To assess the pulmonary ﬁbrosis or lung injury, DNA microarrays has been performed to identify clusters of genes involved in the progression of these pulmonary diseases (Katsuma et al.,

48

K. Fujita et al. / Toxicology 258 (2009) 47–55

2001; McDowell et al., 2003; Kaminski and Rosas, 2006; Studer and Kaminski, 2007). Furthermore, gene expression analysis has been used to elucidate the toxicological effects of nanoparticles (Chen et al., 2006; Chou et al., 2008). We have previously analyzed gene expression proﬁles in rat lungs after intratracheal instillation of ultraﬁne nickel oxide particles (Fujita et al., in press). These gene expression proﬁles corresponded well to the results from conventional methods such as immunohistochemical analysis and bronchoalveolar lavage ﬂuid (BALF) cell analysis. We suggest that gene expression analysis using DNA microarrays can be useful in assessing the inﬂuence of utraﬁne nanoparticles on biological systems. We hereby report the gene expression proﬁles of the rat lung after whole-body inhalation exposure to C60 fullerene in order to assess the inﬂuence of the nanoparticles on molecular events. This study was conducted to examine pulmonary response at 3 days and 1 month post-exposure to C60 fullerene for 6 h a day, for 4 weeks (5 days a week) to assess the short-term effects of the inhalation post-exposure. Nanoparticles have unique physical and chemical properties. They are unstable and tend to agglomerate in aqueous solution. Well-characterized nanoparticles are required for the accurate assessment of toxicity associated with ultraﬁne nanoparticles. The characterization of nanoparticles by robust physicochemical techniques is required to resolve this problem (Oberdörster et al., 2005a,b). In this research, we paid meticulous attention to the characterization of the nanoparticles in aqueous suspensions and the atmospheric aerosols in inhalation system. A number of reports have described the pulmonary toxicity assessments of nickel oxide exposure and animal studies have linked nickel oxide to the development of lung cancer, acute lung injury and inﬂammation (Morimoto et al., 1995; Oyabu et al., 2007; Kawanishi et al., 2002). Given this wealth of information, we examined the effects of C60 fullerene particles on pulmonary responses compared with ultraﬁne nickel oxide particles as a positive control agent. 2. Materials and methods 2.1. Particle characterization Bulk high-purity (>99.5%) C60 fullerene was purchased from Frontier Carbon Corporation (Nanom purple, Fukuoka, Japan). The manufacturer’s speciﬁcations indicated a speciﬁc surface area of 0.92 m2 /g. Bulk C60 fullerene material dispersed in 0.1 mg/ml polyoxyethylene sorbitan monooleate (Tween-80, Wako Pure Chemical Industries, Ltd., Tokyo, Japan) was milled in an agate mortar for 30 min under a nitrogen atmosphere. The milled C60 fullerene material was suspended with zirconium particles (50 ␮m diameter) using a high-performance dispersion machine (UAM-15, Kotobuki Industries Co., Ltd., Tokyo, Japan) and centrifuged at 8000 × g for 60 min. The concentrations were determined by an HPLC system (#1100, Agilent Technologies, Santa Clara, CA). Bulk nano-sized nickel oxide particles used in this study were purchased from Nanostructured and Amorphous Materials Inc. (#4210SD, Houston, TX). The manufacturer’s speciﬁcations indicated an average primary particle size of 10–20 nm, and a speciﬁc surface area of 50–80 m2 /g. Nickel oxide particles dissolved in deionized water (0.5%, w/w) were dispersed by an ultrasonic homogenizer (450W, 90 min at 10 min intervals), and centrifuged at 10,000 × g for 20 min. The supernatant was 1.0 ␮m membrane-ﬁltered. Following the preparations, the particle size of C60 fullerene and nickel oxide were estimated using the laser light diffraction method performed with the Microtrac® UPA150 device (Nikkiso Co., Ltd., Tokyo, Japan). Their structures and diameters were observed by a transmission electron microscope (TEM) at 200 kV (Leo, Oberkochen, Germany). 2.2. Aerosol generation and atmosphere characterization The whole-body exposure system used to expose rats to clean air, C60 fullerene or Uf-NiO aerosols consisted of a pressurized nebulizer and a mist dryer, connected to an exposure chamber (volume: 0.52 m3 ) at a controlled temperature of 21 ◦ C and 56% relative humidity. The size and number concentrations of aerosol particles from the inlet valve and inside of exposure chamber were analyzed in-line using a particle spectrometer consisting of a differential mobility analyzer (DMA) and a condensation particle counter (CPC) (Model 1000XP WPS, MSP Corp., Shoreview, MN). Prior to initiating animal exposure tests, generated aerosols were sampled from different positions in the chamber’s inner space in order to measure the distribution of the aerosol particle size. The results showed that a sufﬁciently uniform aerosol distribution inside the chamber could be obtained. The stability of the C60 fullerene

and Uf-NiO aerosol particle size under a continuous operation of 6 h was conﬁrmed. The C60 fullerene aerosol in the exposure chamber had an average mass concentration of 0.12 mg/m3 (0.5 ± 0.1 mg/m3 : including Tween-80), a particle concentration of 4.1 × 104 particles/cm3 , and an average geometric diameter of 96 nm. The UfNiO particle aerosol in the exposure chamber had an average mass concentration of 0.2 ± 0.1 mg/m3 , a particle concentration of 9.2 × 104 particles/cm3 , and an average geometric diameter of 59 nm. We conﬁrmed that these aerosol particle sizes and concentrations in the chamber were stable for at least 4 weeks (Shimada et al., 2008). 2.3. C60 fullerene and nickel lung burden Left lung tissues exposed to clean air, C60 fullerene or Uf-NiO were individually homogenized and digested via a digester (Multiwave 3000, Anton Paar GmbH, Austria). The concentration of C60 fullerene or nickel in the homogenized tissues was determined by liquid chromatography combined with UV absorptiometry, LCUV (HP1100, Agilent Technologies, Santa Clara, CA) or an inductively coupled argon plasma mass spectrometer, ICP-MS (Agilent Technologies, Santa Clara, CA) respectively. The particle burdens were estimated using the left lung weights. Signiﬁcant differences between time points within each group were assessed by one-way factorial ANOVA. Student’s t-test was used to assess statistical signiﬁcance at P < 0.05. The size of the C60 fullerene and Uf-NiO particles was characterized by a TEM at 200 kV (Leo, Oberkochen, Germany). 2.4. Animals Groups of 9-week-old male Wistar rats (n = 5 per group/time point) purchased from Kyudo Co., Inc. (Kumamoto, Japan) were exposed to clean air (negative control), C60 fullerene or Uf-NiO (positive control) suspension in a whole-body exposure chamber. Rats were exposed to aerosol for 6 h a day, for 4 weeks (5 days a week). Standard chow diet was provided ad libitum during non-exposure hours. After exposure period for 4 weeks, rats were housed within polycarbonate cages at a controlled temperature of 22 ◦ C with a chow diet ad libitum, and dissected at 3 days and 1 month (33 days) post-exposure. Lungs of anesthetized rats were perfused with physiological saline, excised, and used for C60 fullerene or nickel burdens in lungs (n = 4, left lungs), TEM analysis (n = 1, whole lungs) and microarray analysis (n = 4, right lungs). Animal procedures were approved by the University of Occupational and Environmental Health, Japan and the National Institute of Advanced Industrial Science and Technology, Japan Animal Care and Use Committees. 2.5. RNA extraction and DNA microarray The right lungs were homogenized using QIAzol lysis reagent with a Tissue Ruptor (Qiagen, Tokyo, Japan). Total RNA from the homogenates was extracted using the RNeasy Midi kit (Qiagen, Tokyo, Japan) following the manufacturer’s instructions. RNA quality and concentration were determined using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) and a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE). cRNA labeled with ﬂuorescent Cyanine 3-CTP was used for hybridization onto the Whole Rat Genome Oligo Multiplex Microarray slides (#G4131F, Agilent Technologies, Santa Clara, CA) containing approximately 41,000 oligonucleotide probes at 65 ◦ C for 17 h. Hybridized microarray slides were washed according to the manufacturer’s instructions, and were scanned with an Agilent DNA Microarray Scanner (#G2565BA, Agilent Technologies, Santa Clara, CA) at 5 ␮m resolution. The scanned images were analyzed numerically using the Agilent Feature Extraction Software version 9.5.3.1. 2.6. Microarray data analysis Normalized data were analyzed using GeneSpring GX version 7.3.1 software (Agilent Technologies, Santa Clara, CA). In each nanoparticle exposure experiment at the same post-exposure periods, genes with over a 2-fold or less than 0.5fold intensity ratio compared with those of clean air exposure (negative control) were considered as up- or down-regulated, respectively. P-values by one sample Student’s t-test were calculated for each sample in each of the experimental groups. Gene expression data for each of the experimental groups are provided in Supplementary Table 1, and deposited in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/projects/geo/). The differences between the control and the experimental groups were evaluated with the Student’s t-test. Pvalues less than 0.05 were considered signiﬁcant. The Web-based application GOstat (http://gostat.wehi.edu.au/) was used to identify statistically overrepresented Gene Ontology (GO) terms (Beissbarth and Speed, 2004).

3. Results 3.1. Characterization of nanomaterials C60 fullerene particles and the crystal lattice in the supernatant were observed by a TEM (Fig. 1A and B), their diameter

K. Fujita et al. / Toxicology 258 (2009) 47–55

49

Fig. 1. Characterization of C60 fullerene suspension dispersed in Tween-80 and Uf-NiO suspension dispersed in deionized water. (A) Zero-loss imaging of cluster of C60 fullerene particles. (B) Lattice imaging for crystal C60 fullerene particles. (C) Zero-loss imaging of cluster of Uf-NiO particles. (D) Size distribution of Uf-NiO suspension dispersed in deionized water. The histogram and cumulative distribution of particle diameter based on the volume were expressed with a dashed line and a full line, respectively.

was estimated to be approximately 30 nm. The size distribution and crystal structure of the C60 fullerene suspensions were maintained for 3 weeks at room temperature (Endoh and Uchida, 2008). Meanwhile, a fraction of the aggregate formation of nickel oxide particles was also observed by TEM analysis (Fig. 1C). Based on the measurement of the cumulative size distribution per volume, the median diameter was in the 20–30 nm range. Alternatively, the average particle size was 8.3 nm using the Sauter mean diameter (SMD) parameter, deﬁned as the diameter of a sphere that has the same volume/surface area ratio as a particle (Fig. 1D). Most of the nickel oxide particles were stable in suspension as primary particle status, and these physicochemical properties were maintained for 3 weeks at room temperature (data not shown). Thus, we deﬁned the nickel oxide particles as ultraﬁne nickel oxide (UfNiO). The C60 fullerene and Uf-NiO suspensions were utilized for the following animal exposure tests using the whole-body exposure system consisting of a pressurized nebulizer and an exposure chamber. 3.2. Anatomical observations in rats There were no signiﬁcant differences in body, lung, liver, or brain weight in the three groups (n = 5, data not shown). No obvious morphological changes were observed in the clean air or C60 fullerene exposure groups at 3 days and 1 month post-exposure. Nodule-like lesions were observed in animals that were exposed to Uf-NiO particles at both 3 days (n = 2/5) and 1 month (n = 1/5) postexposure (data not shown). No histopathological abnormalities were observed in the liver, kidney, spleen, cerebrum, cerebellum, testis, or nasal cavity tissues in the three groups (n = 5).

MS analyses indicated that C60 fullerene or nickel remained in lung tissues at 3 days post-exposure, and decreased at 1 month postexposure. In the group of rats exposed to clean air, C60 fullerene or nickel were below the detection limit. To validate the results of the quantitative analyses, C60 fullerene or Uf-NiO particles in the lung tissues were observed by TEM analysis. Zero-loss images demonstrated that some particles remained in alveolar epithelial cells at 3 days post-exposure with C60 fullerene, however, the crystal lattice shown in Fig. 1B was not conﬁrmed (Fig. 2A). No particles were identiﬁed in alveolar epithelial cells at 1 month post-exposure with C60 fullerene. In alveolar macrophages at both 3 days and 1 month post-exposure with C60 fullerene, some particles like the ones as mentioned in Fig. 1A were observed (Fig. 2B). Meanwhile, some particles remained in alveolar epithelial cells at 3 days post-exposure with Uf-NiO (Fig. 2C). Nickel was detected from these particles by electron energy-loss spectroscopy (EELS) analysis (Fig. 2D), so these particles were identiﬁed as nickel oxide particles. No nickel oxide particles were identiﬁed in alveolar epithelial cells at 1 month postexposure with Uf-NiO. We also observed some traces in alveolar macrophages at 3 days and 1 month post-exposure with Uf-NiO by zero-loss images, and identiﬁed as nickel oxide particles by EELS analysis (Fig. 2E and F). No nickel oxide particles were observed in bronchial epithelial cells at 3 days and 1 month post-exposure (data not shown). Table 1 C60 fullerene and nickel burden in lung tissues after inhalation exposure. Post-exposure

C60 fullerene

Nickel

3 days

1 month

3 days

1 month

u.l. 3.40 ± 0.37 n.d.

u.l. 1.91 ± 0.24 n.d.

0.13 ± 0.04 n.d. 4.99 ± 0.19

0.08 ± 0.02 n.d. 2.86 ± 0.27

3.3. C60 fullerene and nickel in lung tissues

Control C60 fullerene Uf-NiO

Table 1 shows the time course changes in C60 fullerene or nickel weight in the left lung after whole-body exposure. LC-UV or ICP-

The particle weight per left lung tissue was expressed as mean ± S.E.M. (␮g). C60 detection limit < 0.012 ␮g; Ni detection limit < 0.021 ␮g; u.l., undetectable levels; n.d., not determined.

50

K. Fujita et al. / Toxicology 258 (2009) 47–55

Fig. 2. C60 fullerene and Uf-NiO particles in rat lung tissues. (A) C60 fullerene in alveolar epithelial cells at 3 days post-exposure. (B) C60 fullerene in alveolar macrophage at 1 month post-exposure. (C) Uf-NiO in alveolar epithelial cells at 3 days post-exposure. (E) Uf-NiO in alveolar macrophage at 1 month post-exposure. (D and F) Mapping images obtained by electron energy-loss spectroscopy (EELS) analysis were identiﬁed as nickel oxide particles.

3.4. DNA microarray analysis Using genes that were up-regulated over twofold (P < 0.05), signiﬁcantly overrepresented Gene Ontology categories within the Biological process were clustered (Tables 2 and 3). The results reveal a different pattern of expression of gene ontology categories at 3 days and 1 month between C60 fullerene and Uf-NiO post-exposure. Signiﬁcantly overrepresented GO terms by C60 fullerene exposure at 3 days post-exposure were classiﬁed broadly into 2 groups, the response stimulus (GO: 0050896) and the immune system process (GO: 0002376) (Table 2). Defense response (GO: 0006952), response to external stimulus (GO: 0009605) both branched from the response stimulus, and B cell homeostasis (GO: 0001782), lymphocyte homeostasis (GO: 0002260), leukocyte homeostasis (GO: 0001776) all branched from the immune system process and were all highly signiﬁcant. Subsequently, a number of signiﬁcant GO categories increased following C60 fullerene exposure at 1 month post-exposure. Genes up-regulated by C60 fullerene exposure were found to be signiﬁcantly related with biological regulation (GO: 0065007) and its branches, such as intracellular signaling cascade (GO: 0007242), regulation of biological process (GO: 0050789), etc. Additionally, calcium ion transport (GO: 0006816), which is a lower layer of metal ion transport (GO: 0030001), cation transport (GO: 0006812), or di-, tri-valent inorganic cation transport (GO: 0015674), or response to external stimulus (GO: 0009605), immune system process (GO: 0002376) were also exhibited. A small number of up-regulated genes categorized into inﬂammatory response, C2, Ccl12, Cx3cl1, and Il4ra were identiﬁed (Table 4). The representative genes involved in oxidative stress, apoptosis, and metalloendopeptidase activity were not observed (Tables 5 and 6). Some of the genes associated with immune response were up-regulated at both 3 days and 1 month post-exposures (Table 7); Cd79b and Cd19 are categorized into the B cell receptor signaling pathway (GO: 0050853). Additionally, some genes such as RT1-A3, RT-1CE2, RT1CE10 associated with the immune system process, including major histocompatibility complex (MHC)-mediated immunity were upregulated. Using down-regulated genes, the representative Gene Ontology terms within the Biological process which were signiﬁcantly overrepresented by C60 fullerene exposure at 3 days post-exposure were not identiﬁed (Supplementary Table 2). Apoptosis (GO: 0006915) and the programmed cell death (GO: 0012501) were exhibited; however, the representative genes involved in the categories such as genes encoding caspase were not down-regulated (Table 5). Signiﬁcantly overrepresented GO terms by C60 fullerene exposure at 1 month post-exposure were classiﬁed into the sensory perception

(GO: 0007600), signal transduction (GO: 0007165) and its branches. In deed, a large number of genes encoding predicted member of the olfactory receptor family were identiﬁed in these GO categories (Supplementary Table 1). The representative down-regulated genes involved in inﬂammatory response, oxidative stress, apoptosis, metalloendopeptidase activity, and immune response were not exhibited (Tables 3–7). In contrast, P-values of the signiﬁcantly overrepresented GO terms for Uf-NiO exposure at 3 days and 1 month post-exposure were extremely lower than those of C60 fullerene post-exposure (Table 3). These results suggest that genes up-regulated by UfNiO exposure are remarkably related with certain gene functions as compared with C60 fullerene exposure. Signiﬁcantly overrepresented GO terms for 3 days Uf-NiO post-exposure distinctively identiﬁed GO categories such as inﬂammatory response (GO: 0006954), immune response (GO: 0006955), and chemotaxis (GO: 0006935). Furthermore, lower P-values were detected at 1 month post-exposure. GO categories involved in the inﬂammatory and immune response were signiﬁcantly overrepresented (Table 3). These included various genes representative of those involved in the inﬂammatory response, such as Ccl2 (MCP-1) a monocyte chemoattractant, Ccl3 (MIP-1a), Ccl5 (RANTES), Ccl7, Ccl12, and Ccl17 (Table 4). Remarkably, the Cxcl1 (CINC-1) and Cxcl2 (Mip2) genes, which act as a neutrophil chemoattractant or play a role in the acute phase of the inﬂammatory response were expressed at high levels at 3 days post-exposure. C3 and C4bpa genes that are involved in the innate immune response or function as regulatory proteins for the complement system of the innate immune response exhibited a high-level of induction. Furthermore, Gpx1, Hmox1 (Ho-1), Sod2 that are associated with the oxidative stress response (GO: 0006979) were signiﬁcant up-regulated (Table 5). Among genes involved in apoptosis (GO: 0006915), Lcn2 encoding a member of the lipocalin superfamily with diverse functions such as the regulation of inﬂammatory responses, control of cell growth and development, tissue involution, and apoptosis was signiﬁcantly up-regulated by Uf-NiO exposure. The expression level was also high at 1 month post-exposure. Signiﬁcant high expression levels of Mmp12, encoding a macrophage metalloelastase/matrix metalloproteinase were observed at 3 days and 1 month post-exposure following Uf-NiO exposure (Table 6). On the other hand, the expression of the genes coding for antiproteases, such as Timp1, Timp2, and Timp3 encoding tissue inhibitors of metalloproteinases, were not highly induced. Using down-regulated genes, signiﬁcantly overrepresented GO terms by Uf-NiO exposure at 3 days post-exposure were classiﬁed into the response to stress (GO: 0006950), inﬂammatory response

K. Fujita et al. / Toxicology 258 (2009) 47–55

51

Table 2 Statistically overrepresented GO terms within up-regulated genes by C60 fullerene exposure. Rank

3 days post-exposure

1 month post-exposure

GO ID

GO terms

Number in the group

Number changed

P-value

GO ID

GO terms

1

0050896

3773

24

5.5E−04

0065007

2

0002376

908

11

4.5E−03

0007242

3

0001782

Response to stimulus Immune system process B cell homeostasis

7

2

2.5E−02

0050789

4 5

0006952 0030856

490 10

7 2

3.2E−02 3.2E−02

0030001 0048518

6

0009605

700

8

3.5E−02

0032502

7

0002260

Defense response Regulation of epithelial cell differentiation Response to external stimulus Lymphocyte homeostasis

13

2

4.0E−02

0048856

8

0048872

15

2

4.0E−02

0050794

9

0001776

15

2

4.0E−02

0006812

10

0045638

17

2

4.0E−02

0009653

11

0003008

Homeostasis of number of cells Leukocyte homeostasis Negative regulation of myeloid cell differentiation System process

Biological regulation Intracellular signaling cascade Regulation of biological process Metal ion transport Positive regulation of biological process Developmental process Anatomical structure development Regulation of cellular process Cation transport

2323

14

4.0E−02

0048523

12

0048469

Cell maturation

77

3

4.0E−02

0048522

13

0002474

77

3

4.0E−02

0048519

14

0042221

1970

13

4.0E−02

0009605

15

0045595

182

4

4.0E−02

0016043

16

0045667

20

2

4.0E−02

0002376

17

0009611

471

6

4.0E−02

0006816

18

0048002

87

3

4.4E−02

0015674

19

0030099

88

3

4.4E−02

0006950

20

0021700

Peptide antigen processing and presentation via MHC class I Regulation of osteoblast differentiation Regulation of cell differentiation Regulation of osteoblast differentiation Response to wounding Peptide antigen processing and presentation Myeloid cell differentiation Developmental maturation

90

3

4.4E−02

0006357

(GO: 0006954) and its related (Supplementary Table 3). GO terms overrepresented in down-regulated genes by Uf-NiO exposure at 1 month post-exposure contained the sensory perception (GO: 0007600), signal transduction (GO: 0007165) and its branches. As well as the result of the C60 fullerene exposure, a large number of genes encoding predicted member of the olfactory receptor family were identiﬁed in these GO categories. The representative down-regulated genes involved in inﬂammatory response, oxidative stress, apoptosis, metalloendopeptidase activity, and immune response were not exhibited (Tables 3–7). 4. Discussion In this study, Uf-NiO was used as a positive control agent for C60 fullerene. It has been reported that slight histopathological changes were observed in rats exposed to nickel oxide nanoparticles (Oyabu et al., 2007). Histopathological lesions were observed in rats lungs exposed to conventional nickel oxide in systemic instillation systems (Dunnick et al., 1989). We have previously demonstrated

Number changed

P-value

5974

96

7.9E−10

1840

42

3.2E−09

5285

86

3.2E−09

654 1042

21 27

2.0E−08 1.8E−07

3109

56

1.8E−07

2030

41

5.1E−07

4791

74

1.2E−06

829

22

2.3E−06

1078

26

2.3E−06

998

24

8.6E−06

936

23

8.6E−06

1069

25

9.1E−06

Response to external stimulus

700

19

9.1E−06

Cell organisation and biogenesis Immune system process

3248

53

2.0E−05

908

22

2.0E−05

184

11

3.0E−05

268

13

3.0E−05

1136

25

3.9E−05

430

16

4.6E−05

Anatomical structure morphogenesis Negative regulation of cellular process Positive regulation of cellular process Negative regulation of biological process

Calcium ion transport Di-, tri-valent inorganic cation transport Response to stress Regulation of transcription from Pol II promoter

Number in the group

that polymorphonuclear leukocytes and inﬂammatory lesions were increased in rat lungs after intratracheal instillation of Uf-NiO particles (Fujita et al., in press). In the present study, DNA microarray analysis indicated that up-regulated genes by Uf-NiO inhalation exposure were overrepresented in gene ontology categories such as the inﬂammatory response and immune system process (Table 3). Also, high expression levels of individual genes involved in the inﬂammatory response, oxidative stress, and Mmp12 were exhibited for Uf-NiO post-exposure (Tables 4–6). Hence, the gene expression proﬁle data can lead to phenotypical and toxicological relevant effects of Uf-NiO inhalation. If that is true, C60 fullerene might not have a severe pulmonary toxicity under this experimental condition. Because genes involved in the inﬂammatory response, oxidative stress response, apoptosis, metalloendopeptidase activity were not up-regulated following C60 fullerene inhalation (Tables 4–6). No in vivo studies to date have reported serious pulmonary toxicity induced by C60 fullerene particles. An assessment of toxicity resulting from inhalation exposure to C60 fullerene nanoparticles and microparticles determined min-

52

K. Fujita et al. / Toxicology 258 (2009) 47–55

Table 3 Statistically overrepresented GO terms within up-regulated genes by Uf-NiO exposure. Rank

3 days post-exposure GO ID

GO terms

1

0009605

2

0009611

3

0006954

Response to external stimulus Response to wounding Inﬂammatory response

4

0002376

5

0006955

Immune system process Immune response

6

0050896

7

0006935

8 9

1 month post-exposure Number changed

P-value

GO ID

GO terms

700

24

1.2E−13

0009605

471

18

7.1E−11

0009611

284

15

7.1E−11

0048856

908

23

8.0E−11

0065008

701

20

3.2E−10

0032502

Response to stimulus Chemotaxis

3773

39

4.6E−10

0065007

110

10

1.5E−09

0048518

0042330 0007626

Taxis Locomotory behavior

110 170

10 11

1.5E−09 4.6E−09

0007610 0048519

10

0006952

Defense response

490

16

4.6E−09

0048869

11

0065008

1008

21

1.3E−08

0030154

12

0007610

Regulation of biological quality Behavior

Response to external stimulus Response to wounding Anatomical structure development Regulation of biological quality Developmental process Biological regulation Positive regulation of biological process Behavior Negative regulation of biological process Cellular developmental process Cell differentiation

332

13

3.2E−08

0048731

13

0048523

998

20

6.4E−08

0006950

14

0051179

4473

40

8.7E−08

0048523

15

0006629

817

18

9.8E−08

0048522

16

0048518

1042

20

1.1E−07

17

0048519

1069

20

18

0044255

657

19

0048545

20

0042221

Negative regulation of cellular process Localization Lipid metabolic process Positive regulation of biological process Negative regulation of biological process Cellular lipid metabolic process Response to steroid hormone stimulus Response to chemical stimulus

Number in the group

Number changed

t-value

700

56

3.2E−33

471

40

1.6E−25

2030

99

1.7E−25

1008

63

2.8E−25

3109

129

4.3E−24

5974

200

5.7E−23

1042

62

6.4E−23

332 1069

30 61

4.7E−21 5.9E−21

1689

82

5.9E−21

1689

82

5.9E−21

1727

83

8.1E−21

1136

63

1.2E−20

998

57

1.3E−19

936

54

6.7E−19

0006928

Negative regulation of cellular process Positive regulation of cellular process Cell motility

401

32

6.7E−19

1.6E−07

0051674

Localization of cell

401

32

6.7E−19

16

1.8E−07

0007275

2232

94

7.3E−18

100

8

2.2E−07

0050789

5285

172

8.6E−18

1970

23

3.2E−07

0002376

Multicellular organismal development Regulation of biological process Immune system process

908

51

5.5E−17

imal changes in the toxicological endpoints (Baker et al., 2008). The suspensions of C60 fullerene in water had little or no difference in rat lungs intratracheally instilled with C60 fullerene particles when compared to control samples (Sayes et al., 2007). However, there is a possibility that the low exposure concentration of C60 resulted in the gene expression proﬁles in this study. The no-observed-adverse-effect level (NOAEL) of C60 fullerene for the pulmonary toxicity might be higher. To elucidate the pulmonary toxicity of C60 fullerene, a study of high-dose exposure is needed. In this study, one of signiﬁcantly overrepresented GO categories by C60 fullerene exposure at 3 days and 1 month post-exposures were classiﬁed into the immune system process (GO: 0002376) (Table 2). In individual gene expression results, a member of the B cell antigen receptor (BCR) complex assembled on the cell surface, Cd79b (Ig␤) gene and BCR signaling regulated membrane coreceptors, CD19 gene were up-regulated at 1 month post-exposure (Table 7). Some of the genes involved in RT1 genes, the rat major histocompatibility complex (MHC), were up-regulated by C60 fullerene exposure. Additionally, interleukin 4 receptor gene Il4ra was upregulated at 1 month post-exposure (Table 4). Interleukin 4 is

System development Response to stress

Number in the group

a multifunctional cytokine that plays a critical role in the regulation of immune responses (Nelms et al., 1999). We have no control experiment whether this receptor is up-regulated on the surface of the cells; however, these results suggest that immune system may function under the C60 fullerene exposure condition in which genes involved in the inﬂammatory response are not induced. The immunological properties of engineered nanomaterials have been reviewed (Dobrovolskaia and McNeil, 2007). And it has been reported that C60 fullerene particles may inhibit the anaphylaxis (Ryan et al., 2007). C60 fullerene particles may stimulate and/or suppress the immune responses after exposure. Mitchell et al. demonstrated that carbon-based particle, multiwalled carbon nanotubes (MWCNT) by whole-body inhalation exposure did not result in signiﬁcant lung inﬂammation or tissue damage, but caused systemic immune function alterations (Mitchell et al., 2007). An interesting point here is that no changes in immune function related gene expression were observed in lung; however, interleukin 10 mRNA levels were increased in spleen. C60 fullerene may also be potentially bypassing pulmonary defense mechanisms and reaching circulation. So far, little is known about the effects of C60 fullerene on the immune system (immunotoxicity) in the in vivo

K. Fujita et al. / Toxicology 258 (2009) 47–55

53

Table 4 Expressed genes associated with the inﬂammatory response. Genbank

Gene name

NM 172222 NM 016994 NM 031504 NM 012516 NM 053619 NM 031530 NM 013025 NM 053858 NM 031116 NM 001012357 XM 213425 NM 057151 NM 019233 NM 001008513 NM 020542 NM 021866 NM 134455 NM 030845 NM 053647 NM 138522 NM 145672 NM 139089 AF217564 NM 017019 NM 031512 NM 133380 NM 017183 AY077842

C2 C3 C4a C4bpa C5ar1 Ccl2/MCP-1 Ccl3/MIP-1a Ccl4/Mip1-b Ccl5/RANTES Ccl9 Ccl12 Ccl17 Ccl20 Ccl21b Ccr1 Ccr2 Cx3cl1 Cxcl1/CINC-1 Cxcl2/Mip-2 Cxcl3/Cinc-2 Cxcl9/Mig Cxcl10/IP-10 Cxcl12 Il1a Il1b Il4ra Il8rb Il18

C60 fullerene

Uf-NiO

Description

3 days

1 month

3 days

1 month

0.9 0.9 0.9 0.9 1.0 1.4 1.4 1.5 1.0 0.7 1.7 0.6 0.8 0.6 0.9 1.4 1.0 1.7 1.6 0.9 1.1 1.0 1.0 0.8 0.8 0.6 0.7 1.1

2.0 1.7 1.6 1.1 1.1 1.3 1.2 1.2 1.2 1.2 2.2 1.6 1.1 1.5 1.4 1.3 2.2 0.9 1.2 1.5 1.1 0.9 1.2 1.1 1.5 2.1 0.7 1.5

1.0 3.3 0.7 3.1 1.4 5.1 2.6 1.7 0.8 1.7 4.0 2.3 1.4 0.8 0.6 1.5 1.2 6.2 8.9 1.2 0.8 0.7 1.3 0.9 1.1 0.4 0.9 1.4

3.1 2.4 2.3 2.7 2.1 1.8 1.8 1.2 1.2 2.2 2.8 1.5 1.0 1.3 2.3 1.0 2.7 1.4 1.4 2.0 0.5 0.9 1.4 1.0 1.3 2.4 0.6 2.5

Complement component 2 Complement component 3 Complement component 4a Complement component 4 binding protein Complement component 5a receptor 1 Chemokine (C-C motif) ligand 2 Chemokine (C-C motif) ligand 3 Chemokine (C-C motif) ligand 4 Chemokine (C-C motif) ligand 5 Chemokine (C-C motif) ligand 9 Chemokine (C-C motif) ligand 12 Chemokine (C-C motif) ligand 17 Chemokine (C-C motif) ligand 20 Chemokine (C-C motif) ligand 21b Chemokine (C-C motif) receptor 1 Chemokine (C-C motif) receptor 2 Chemokine (C-X3-C motif) ligand 1 Chemokine (C-X-C motif) ligand 1 Chemokine (C-X-C motif) ligand 2 Chemokine (C-X-C motif) ligand 3 Chemokine (C-X-C motif) ligand 9 Chemokine (C-X-C motif) ligand 10 Chemokine (C-X-C motif) ligand 12 Interleukin 1 alpha Interleukin 1 beta Interleukin 4 receptor Interleukin 8 receptor, beta Interleukin 18

Numerical values represent gene expression fold change compared to control levels. Table 5 Expressed genes associated with response to oxidative stress and apoptosis. Genbank

Gene name

C60 fullerene 3 days

1 month

3 days

1 month

NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM

Cat Gpx1 Gpx3 Hmox1/Ho-1 Sod1 Sod2 Txnrd1 Casp1/Ice Casp2 Casp3/Lice Casp4 Casp7 Casp8 Casp12 Lcn2 p53/Tp53

1.1 0.7 1.0 0.9 0.7 1.1 0.7 1.2 1.2 0.8 1.1 0.8 1.4 0.9 0.9 0.6

1.2 1.5 2.7 1.5 1.4 0.9 1.4 1.0 1.1 1.0 1.2 1.0 1.2 1.1 1.7 1.1

1.2 1.0 0.6 2.5 1.0 1.9 1.2 1.1 1.0 0.9 1.2 0.9 0.8 0.9 8.6 0.6

1.3 2.0 2.2 3.2 1.5 1.3 1.8 1.0 1.0 1.0 1.4 1.1 1.4 1.1 5.9 1.1

012520 030826 022525 012580 017050 017051 031614 012762 022522 012922 053736 022260 022277 130422 130741 030989

Uf-NiO

Description

Catalase Glutathione peroxidase 1 Glutathione peroxidase 3 Heme oxygenase (decycling) 1 Superoxide dismutase 1 Superoxide dismutase 2 Thioredoxin reductase 1 Caspase 1 Caspase 2 Caspase 3 Caspase 4 Caspase 7 Caspase 8 Caspase 12 Lipocalin 2 Tumor protein p53

Numerical values represent gene expression fold change compared to control levels. Table 6 Expressed genes associated with metalloendopeptidase activity. Genbank

NM 031054 NM 031055 NM 012980 NM 053963 M60616 NM 031056 NM 080776 NM 053606 NM 031757 NM 053819 NM 021989 NM 012886

Gene name

Mmp2 Mmp9 Mmp11 Mmp12 Mmp13 Mmp14 Mmp16 Mmp23 Mmp24 Timp1 Timp2 Timp3

C60 fullerene

Uf-NiO

Description

3 days

1 month

3 days

1 month

0.9 0.9 0.8 1.2 1.0 1.0 0.7 0.7 0.5 0.7 0.6 0.9

1.4 1.3 1.6 1.1 1.3 1.2 1.0 1.6 1.2 1.3 1.3 1.5

1.0 0.9 0.6 76.1 1.0 1.3 0.6 0.6 0.6 0.8 0.9 0.7

1.2 1.5 1.8 11.8 1.2 1.7 0.9 1.4 1.1 1.2 1.4 1.6

Numerical values represent gene expression fold change compared to control levels.

Matrix metallopeptidase 2 Matrix metallopeptidase 9 Matrix metalloproteinase 11 Matrix metallopeptidase 12 Matrix metallopeptidase 13 Matrix metalloproteinase 14 Matrix metalloproteinase 16 Matrix metalloproteinase 23 Matrix metallopeptidase 24 Tissue inhibitor of metalloproteinase 1 Tissue inhibitor of metalloproteinase 2 Tissue inhibitor of metalloproteinase 3

54

K. Fujita et al. / Toxicology 258 (2009) 47–55

Table 7 Expressed genes associated with the immune response. Genbank

Gene name

3 days

1 month

3 days

1 month

NM NM NM NM

Cd79b Blnk Btk Fos

1.3 1.1 1.1 1.2

2.1 1.5 1.4 3.8

0.8 1.1 1.3 0.8

1.7 1.5 2.0 2.0

NM 021836 NM 030857 XM 344961 NM 012758 NM 017168 NM 001002821 NM 012645 NM 001008829 NM 001008830 NM 001008840 NM 001008841 NR 002155

Junb Lyn Cd19 Syk Plcg2 RT1-T18 RT1-Aw2 RT1A-2 RT1-A3 RT1-CE2 RT1-CE3 RT1-CE6

1.0 0.8 1.8 1.2 1.1 6.4 1.3 0.9 1.4 1.0 0.8 3.6

2.6 1.3 2.1 1.7 1.9 3.4 1.9 2.0 4.9 2.1 2.1 2.6

1.0 1.1 0.9 0.9 0.7 5.5 1.4 1.2 1.7 1.2 1.2 2.4

2.4 1.5 2.0 2.3 1.9 1.1 1.7 1.5 1.9 1.8 2.1 1.1

NM 001008833 NM 001004084 NM 001008846

RT1-CE10 RT1-Bb RT1-DOb

3.4 0.7 1.1

5.8 2.3 2.5

2.2 0.8 0.8

0.9 2.1 2.0

133533 001025767 001007798 022197

C60 fullerene

Uf-NiO

Description

B-cell-speciﬁc membrane protein B-cell linker (Blnk) Putative protein tyrosine kinase An immediate early gene encoding a nuclear protein involved in signal transduction Transcription factor; involved in transcriptional regulation Src-related tyrosine kinase CD19 antigen (predicted) Spleen tyrosine kinase Has phosphoinositide-speciﬁc phospholipase C activity Member of a family of MHC class I-b genes A class Ib gene of the rat major histocompatibility complex RT1 class Ia RT1 class I, A3 RT1 class I, CE2 RT1 class I, CE3 Predicted gene deduced from the genomic sequence of the major histocompatibility complex RT1-CE10 protein RT1 class II Histocompatibility 2, O region beta locus

Numerical values represent gene expression fold change compared to control levels.

setting. Further investigations are needed to elucidate the immune mechanisms responsible. In summary, we used oligonucleotide microarrays to identify and proﬁle the expression of genes in rat lung after wholebody inhalation exposure to C60 fullerene and Uf-NiO particles. In response to C60 fullerene exposure for 4 weeks, there were a small number of highly up-regulated genes associated with the inﬂammatory response, oxidative stress, apoptosis, and metalloendopeptidase activity for 1 month post-exposure. These results were quite different from those of Uf-NiO particles, suggesting that C60 fullerene particles did not induce signiﬁcant inﬂammation and tissue injury for the inhalation exposure period. Meanwhile, some genes associated with the immune system were up-regulated by C60 fullerene particles. Overall, we suggest that C60 fullerene might not have a severe pulmonary toxicity under this experimental condition. Conﬂict of interest The authors declare that there are no conﬂicts of interest. Acknowledgment This research was funded by New Energy and Industrial Technology Development Organization of Japan (NEDO) Grant “Evaluating risks associated with manufactured nanomaterials (P06041)”. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tox.2009.01.005. References Baker, G.L., Gupta, A., Clark, M.L., Valenzuela, B.R., Staska, L.M., Harbo, S.J., Pierce, J.T., Dill, J.A., 2008. Inhalation toxicity and lung toxicokinetics of C60 fullerene nanoparticles and microparticles. Toxicol. Sci. 101, 122–131. Beissbarth, T., Speed, T.P., 2004. GOstat: ﬁnd statistically overrepresented Gene Ontologies within a group of genes. Bioinformatics 12, 1464–1465. Chen, H.W., Su, S.F., Chien, C.T., Lin, W.H., Yu, S.L., Chou, C.C., Chen, J.J., Yang, P.C., 2006. Titanium dioxide nanoparticles induce emphysema-like lung injury in mice. FASEB J. 20, 2393–2395.

Chou, C.C., Hsiao, H.Y., Hong, Q.S., Chen, C.H., Peng, Y.W., Chen, H.W., Yang, P.C., 2008. Single-walled carbon nanotubes can induce pulmonary injury in mouse model. Nano Lett. 8, 437–445. Dobrovolskaia, M.A., McNeil, S.E., 2007. Immunological properties of engineered nanomaterials. Nat. Nanotechnol. 2, 469–478. Dunnick, J.K., Elwell, M.R., Benson, J.M., Hobbs, C.H., Hahn, F.F., Haly, P.J., Cheng, Y.S., Eidson, A.F., 1989. Lung toxicity after 13-week inhalation exposure to nickel oxide, nickel subsulﬁde, or nickel sulfate hexahydrate in F344/N rats and B6C3F1 mice. Fundam. Appl. Toxicol. 12, 584–594. Endoh, S., Uchida, K., 2008. Preparation of suspension of nano-sized particle for toxicology test. In: Proceeding of NEDO-AIST-OECD international symposium on the risk assessment of manufactured nanomaterials, pp. 36–40. Fujita, K., Morimoto, Y., Ogami, A., Tanaka, I., Endoh, S., Uchida, K., Tao, H., Akasaka, M., Inada, M., Yamamoto, K., Fukui, H., Hayakawa, M., Horie, M., Saito, Y., Yoshida, Y., Iwahashi, H., Niki, E., Nakanishi, J., in press. A gene expression proﬁling approach to study the inﬂuence of ultraﬁne particles on rat lungs. In: Kim, J.Y., Platt, U., Gu, M.B., Iwahashi, H. (Eds.), Atmospheric and Biological Environmental Monitoring. Springer-Verlag GmbH, Netherlands. Grassian, V.H., O’shaughnessy, P.T., Adamcakova-Dodd, A., Pettibone, J.M., Thorne, P.S., 2007. Inhalation exposure study of titanium dioxide nanoparticles with a primary particle size of 2 to 5 nm. Environ. Health Perspect. 115, 397–402. Kaminski, N., Rosas, I.O., 2006. Gene expression proﬁling as a window into idiopathic pulmonary ﬁbrosis pathogenesis: can we identify the right target genes? Proc. Am. Thorac. Soc. 3, 339–344. Katsuma, S., Nishi, K., Tanigawara, K., Ikawa, H., Shiojima, S., Takagaki, K., Kaminishi, Y., Suzuki, Y., Hirasawa, A., Ohgi, T., Yano, J., Murakami, Y., Tsujimoto, G., 2001. Molecular monitoring of bleomycin-induced pulmonary ﬁbrosis by cDNA microarray-based gene expression proﬁling. Biochem. Biophys. Res. Commun. 288, 747–751. Kawanishi, S., Oikawa, S., Inoue, S., Nishino, K., 2002. Distinct mechanisms of oxidative DNA damage induced by carcinogenic nickel subsulﬁde and nickel oxides. Environ. Health Perspect. 110, 789–791. McDowell, S.A., Gammon, K., Zingarelli, B., Bachurski, C.J., Aronow, B.J., Prows, D.R., Leikauf, G.D., 2003. Inhibition of nitric oxide restores surfactant gene expression following nickel-induced acute lung injury. Am. J. Respir. Cell Mol. Biol. 28, 188–198. Mitchell, L.A., Gao, J., Wal, R.V., Gigliotti, A., Burchiel, S.W., McDonald, J.D., 2007. Pulmonary and systemic immune response to inhaled multiwalled carbon nanotubes. Toxicol. Sci. 100, 203–214. Morimoto, Y., Nambu, Z., Tanaka, I., Higashi, T., Yamato, H., Hori, H., Cho, S., Kido, M., 1995. Effects of nickel oxide on the production of tumor necrosis factor by alveolar macro-phages of rats. Biol. Trace Elem. Res. 48, 287–296. Nelms, K., Keegan, A.D., Zamorano, J., Ryan, J.J., Paul, W.E., 1999. The IL-4 receptor: signaling mechanisms and biologic functions. Annu. Rev. Immunol. 17, 701–738. Oberdörster, G., Maynard, A., Donaldson, K., Castranova, V., Fitzpatrick, J., Ausman, K., Carter, J., Karn, B., Kreyling, W., Lai, D., Olin, S., Monteiro-Riviere, N., Warheit, D., Yang, H., ILSI Research Foundation/Risk Science Institute Nanomaterial Toxicity Screening Working Group, 2005a. Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy. Part. Fibre Toxicol. 2, 8. Oberdörster, G., Oberdörster, E., Oberdörster, J., 2005b. Nanotoxicology: an emerging discipline evolving from studies of ultraﬁne particles. Environ. Health Perspect. 113, 823–839.

K. Fujita et al. / Toxicology 258 (2009) 47–55 Oyabu, T., Ogami, A., Morimoto, Y., Shimada, M., Lenggoro, W., Okuyama, K., Tanaka, I., 2007. Biopersistence of inhaled nickel oxide nanoparticles in rat lung. Inhal. Toxicol. 19, 55–58. Ryan, J.J., Bateman, H.R., Stover, A., Gomez, G., Norton, S.K., Zhao, W., Schwartz, L.B., Lenk, R., Kepley, C.L., 2007. Fullerene nanomaterials inhibit the allergic response. J. Immunol. 179, 665–667. Sayes, C.M., Marchione, A.A., Reed, K.L., Warheit, D.B., 2007. Comparative pulmonary toxicity assessments of C60 water suspensions in rats: few differences in fullerene toxicity in vivo in contrast to in vitro proﬁles. Nano Lett. 7, 399–406. Shimada, M., Wang, W., Okuyama, K., 2008. Particle dispersion method for inhalation experiment. In: Proceeding of NEDO-AIST-OECD international symposium on the risk assessment of manufactured nanomaterials, pp. 78–82.

55

Stone, V., Johnston, H., Clift, M.J., 2007. Air pollution, ultraﬁne and nanoparticle toxicology: cellular and molecular interactions. IEEE Trans. Nanobiosci. 6, 331– 340. Studer, S.M., Kaminski, N., 2007. Towards systems biology of human pulmonary ﬁbrosis. Proc. Am. Thorac. Soc. 4, 85–91. Warheit, D.B., Laurence, B.R., Reed, K.L., Roach, D.H., Reynolds, G.A., Webb, T.R., 2004. Comparative pulmonary toxicity assessment of single-wall carbon nanotubes in rats. Toxicol. Sci. 77, 117–125. Warheit, D.B., Webb, T.R., Reed, K.L., Frerichs, S., Sayes, C.M., 2007. Pulmonary toxicity study in rats with three forms of ultraﬁne-TiO2 particles: differential responses related to surface properties. Toxicology 30, 90–104.

Gene Expression Changes in the Motor Cortex Mediating ... - PLOS